A hierarchical model for assembly of eukaryotic 60S ribosomal subunit domains.

Despite having high-resolution structures for eukaryotic large ribosomal subunits, it remained unclear how these ribonucleoprotein complexes are constructed in living cells. Nevertheless, knowing where ribosomal proteins interact with ribosomal RNA (rRNA) provides a strategic platform to investigate the connection between spatial and temporal aspects of 60S subunit biogenesis. We previously found that the function of individual yeast large subunit ribosomal proteins (RPLs) in precursor rRNA (pre-rRNA) processing correlates with their location in the structure of mature 60S subunits. This observation suggested that there is an order by which 60S subunits are formed. To test this model, we used proteomic approaches to assay changes in the levels of ribosomal proteins and assembly factors in preribosomes when RPLs functioning in early, middle, and late steps of pre-60S assembly are depleted. Our results demonstrate that structural domains of eukaryotic 60S ribosomal subunits are formed in a hierarchical fashion. Assembly begins at the convex solvent side, followed by the polypeptide exit tunnel, the intersubunit side, and finally the central protuberance. This model provides an initial paradigm for the sequential assembly of eukaryotic 60S subunits. Our results reveal striking differences and similarities between assembly of bacterial and eukaryotic large ribosomal subunits, providing insights into how these RNA-protein particles evolved.

Quantification of trans-synaptic protein alignment: A data analysis case for single-molecule localization microscopy.

Nanoscale distribution of proteins and their relative positioning within a defined subcellular region are key to their physiological functions. Thanks to the super-resolution imaging methods, especially single-molecule localization microscopy (SMLM), mapping the three-dimensional distribution of multiple proteins has been easier and more efficient than ever. Nevertheless, in spite of the many tools available for efficient localization detection and image rendering, it has been a challenge to quantitatively analyze the 3D distribution and relative positioning of proteins in these SMLM data. Here, using heterogeneously distributed synaptic proteins as examples, we describe in detail a series of analytical methods including detection of nanoscale density clusters, quantification of the trans-synaptic alignment between these protein densities, and automatic en face projection and averaging. These analyses were performed within customized Matlab routines and we make the full scripts available. The concepts behind these analytical methods and the scripts can be adapted for quantitative analysis of spatial organization of other macromolecular complexes.

Apolipoprotein E binds to and reduces serum levels of DNA-mimicking, pyrrolated proteins.

Lysine N-pyrrolation, converting lysine residues to Nϵ-pyrrole-l-lysine, is a recently discovered post-translational modification. This naturally occurring reaction confers electrochemical properties onto proteins that potentially produce an electrical mimic to DNA and result in specificity toward DNA-binding molecules such as anti-DNA autoantibodies. The discovery of this unique covalent protein modification provides a rationale for establishing the molecular mechanism and broad functional significance of the formation and regulation of Nϵ-pyrrole-l-lysine-containing proteins. In this study, we used microbeads coupled to pyrrolated or nonpyrrolated protein to screen for binding activities of human serum-resident nonimmunoglobin proteins to the pyrrolated proteins. This screen identified apolipoprotein E (apoE) as a protein that innately binds the DNA-mimicking proteins in serum. Using an array of biochemical assays, we observed that the pyrrolated proteins bind to the N-terminal domain of apoE and that oligomeric apoE binds these proteins better than does monomeric apoE. Employing surface plasmon resonance and confocal microscopy, we further observed that apoE deficiency leads to significant accumulation of pyrrolated serum albumin and is associated with an enhanced immune response. These results, along with the observation that apoE facilitates the binding of pyrrolated proteins to cells, suggest that apoE may contribute to the clearance of pyrrolated serum proteins. Our findings uncover apoE as a binding target of pyrrolated proteins, providing a key link connecting covalent protein modification, lipoprotein metabolism, and innate immunity.

Sequence specificity despite intrinsic disorder: How a disease-associated Val/Met polymorphism rearranges tertiary interactions in a long disordered protein.

The role of electrostatic interactions and mutations that change charge states in intrinsically disordered proteins (IDPs) is well-established, but many disease-associated mutations in IDPs are charge-neutral. The Val66Met single nucleotide polymorphism (SNP) in precursor brain-derived neurotrophic factor (BDNF) is one of the earliest SNPs to be associated with neuropsychiatric disorders, and the underlying molecular mechanism is unknown. Here we report on over 250 µs of fully-atomistic, explicit solvent, temperature replica-exchange molecular dynamics (MD) simulations of the 91 residue BDNF prodomain, for both the V66 and M66 sequence. The simulations were able to correctly reproduce the location of both local and non-local secondary structure changes due to the Val66Met mutation, when compared with NMR spectroscopy. We find that the change in local structure is mediated via entropic and sequence specific effects. We developed a hierarchical sequence-based framework for analysis and conceptualization, which first identifies "blobs" of 4-15 residues representing local globular regions or linkers. We use this framework within a novel test for enrichment of higher-order (tertiary) structure in disordered proteins; the size and shape of each blob is extracted from MD simulation of the real protein (RP), and used to parameterize a self-avoiding heterogenous polymer (SAHP). The SAHP version of the BDNF prodomain suggested a protein segmented into three regions, with a central long, highly disordered polyampholyte linker separating two globular regions. This effective segmentation was also observed in full simulations of the RP, but the Val66Met substitution significantly increased interactions across the linker, as well as the number of participating residues. The Val66Met substitution replaces ß-bridging between V66 and V94 (on either side of the linker) with specific side-chain interactions between M66 and M95. The protein backbone in the vicinity of M95 is then free to form ß-bridges with residues 31-41 near the N-terminus, which condenses the protein. A significant role for Met/Met interactions is consistent with previously-observed non-local effects of the Val66Met SNP, as well as established interactions between the Met66 sequence and a Met-rich receptor that initiates neuronal growth cone retraction.

The BCL-2 protein family: opposing activities that mediate cell death.

BCL-2 family proteins, which have either pro- or anti-apoptotic activities, have been studied intensively for the past decade owing to their importance in the regulation of apoptosis, tumorigenesis and cellular responses to anti-cancer therapy. They control the point of no return for clonogenic cell survival and thereby affect tumorigenesis and host-pathogen interactions and regulate animal development. Recent structural, phylogenetic and biological analyses, however, suggest the need for some reconsideration of the accepted organizational principles of the family and how the family members interact with one another during programmed cell death. Although these insights into interactions among BCL-2 family proteins reveal how these proteins are regulated, a unifying hypothesis for the mechanisms they use to activate caspases remains elusive.

Argonaute proteins: key players in RNA silencing.

During the past decade, small non-coding RNAs have rapidly emerged as important contributors to gene regulation. To carry out their biological functions, these small RNAs require a unique class of proteins called Argonautes. The discovery and our comprehension of this highly conserved protein family is closely linked to the study of RNA-based gene silencing mechanisms. With their functional domains, Argonaute proteins can bind small non-coding RNAs and control protein synthesis, affect messenger RNA stability and even participate in the production of a new class of small RNAs, Piwi-interacting RNAs.

Two-site recognition of phosphatidylinositol 3-phosphate by PROPPINs in autophagy.

Macroautophagy is essential to cell survival during starvation and proceeds by the growth of a double-membraned phagophore, which engulfs cytosol and other substrates. The synthesis and recognition of the lipid phosphatidylinositol 3-phosphate, PI(3)P, is essential for autophagy. The key autophagic PI(3)P sensors, which are conserved from yeast to humans, belong to the PROPPIN family. Here we report the crystal structure of the yeast PROPPIN Hsv2. The structure consists of a seven-bladed ß-propeller and, unexpectedly, contains two pseudo-equivalent PI(3)P binding sites on blades 5 and 6. These two sites both contribute to membrane binding in vitro and are collectively required for full autophagic function in yeast. These sites function in concert with membrane binding by a hydrophobic loop in blade 6, explaining the specificity of the PROPPINs for membrane-bound PI(3)P. These observations thus provide a structural and mechanistic framework for one of the conserved central molecular recognition events in autophagy.

The HARP-like domain-containing protein AH2/ZRANB3 binds to PCNA and participates in cellular response to replication stress.

Proteins with annealing activity are newly identified ATP-dependent motors that can rewind RPA-coated complementary single-stranded DNA bubbles. AH2 (annealing helicase 2, also named as ZRANB3) is the second protein with annealing activity, the function of which is still unknown. Here, we report that AH2 is recruited to stalled replication forks and that cells depleted of AH2 are hypersensitive to replication stresses. Furthermore, AH2 binds to PCNA, which is crucial for its function at stalled replication forks. Interestingly, we identified a HARP-like (HPL) domain in AH2 that is indispensible for its annealing activity in vitro and its function in vivo. Moreover, searching of HPL domain in SNF2 family of proteins led to the identification of SMARCA1 and RAD54L, both of which possess annealing activity. Thus, this study not only demonstrates the in vivo functions of AH2, but also reveals a common feature of this new subfamily of proteins with annealing activity.

Structure of a glomulin-RBX1-CUL1 complex: inhibition of a RING E3 ligase through masking of its E2-binding surface.

The approximately 300 human cullin-RING ligases (CRLs) are multisubunit E3s in which a RING protein, either RBX1 or RBX2, recruits an E2 to catalyze ubiquitination. RBX1-containing CRLs also can bind Glomulin (GLMN), which binds RBX1's RING domain, regulates the RBX1-CUL1-containing SCF(FBW7) complex, and is disrupted in the disease Glomuvenous Malformation. Here we report the crystal structure of a complex between GLMN, RBX1, and a fragment of CUL1. Structural and biochemical analyses reveal that GLMN adopts a HEAT-like repeat fold that tightly binds the E2-interacting surface of RBX1, inhibiting CRL-mediated chain formation by the E2 CDC34. The structure explains the basis for GLMN's selectivity toward RBX1 over RBX2, and how disease-associated mutations disrupt GLMN-RBX1 interactions. Our study reveals a mechanism for RING E3 ligase regulation, whereby an inhibitor blocks E2 access, and raises the possibility that other E3s are likewise controlled by cellular proteins that mask E2-binding surfaces to mediate inhibition.

The tertiary structure of γc cytokines dictates receptor sharing.

The γc family of cytokines comprising interleukin-2 (IL-2), IL-4, IL-7, IL-9, IL-15 and IL-2 is an important group of 4-helix bundle cytokines that signals through receptors incorporating the common gamma chain (γc). These cytokines are involved in lymphocyte biology and their specific functions are contingent on binding to cognate receptor chains. Here, we examined the structural relationships between γc cytokines, aiming to understand the basis for receptor chain usage and sharing. To that end, we obtained tertiary structures of human and mouse γc cytokines plus two other related cytokines, IL-13 and TSLP, which share receptors with IL-4 and IL-7, respectively. Subsequently, we compared the cytokine 3D-structures introducing a structural similarity score that grouped γc cytokines in a manner that mirrored the relationships dictated by receptor sharing. Unlike previously thought, we identified that IL-9 is more closely related to IL-2 and IL-15 than to IL-7, which is actually the most distant member of the γc family of cytokines. Moreover, we found that all the members of the γc family of cytokines share the topology of short-chain 4-helix bundle cytokines but IL-7 that with TSLP has the topology of long-chain 4-helix bundle cytokines. We also carried out Maximun-Likehood and Bayesian phylogenetic analyses that supported these results at the amino acid sequence level. Overall, our findings are of paramount relevance to understand receptor sharing among γc cytokines and can lead to the discovery of new cytokine receptor partners.

CARD-mediated autoinhibition of cIAP1's E3 ligase activity suppresses cell proliferation and migration.

E3 ligases mediate the covalent attachment of ubiquitin to target proteins thereby enabling ubiquitin-dependent signaling. Unraveling how E3 ligases are regulated is important because miscontrolled ubiquitylation can lead to disease. Cellular inhibitor of apoptosis (cIAP) proteins are E3 ligases that modulate diverse biological processes such as cell survival, proliferation, and migration. Here, we have solved the structure of the caspase recruitment domain (CARD) of cIAP1 and identified that it is required for cIAP1 autoregulation. We demonstrate that the CARD inhibits activation of cIAP1's E3 activity by preventing RING dimerization, E2 binding, and E2 activation. Moreover, we show that the CARD is required to suppress cell proliferation and migration. Further, CARD-mediated autoregulation is also necessary to maximally suppress caspase-8-dependent apoptosis and vascular tree degeneration in vivo. Taken together, our data reveal mechanisms by which the E3 ligase activity of cIAP1 is controlled, and how its deregulation impacts on cell proliferation, migration and cell survival.

Dimerization-induced allostery in protein kinase regulation.

The ability of protein kinases to switch between inactive and active states is critical to control the outputs of cellular signaling pathways. In several protein kinases, the conformation of helix αC is a key hub on which regulatory inputs converge to induce catalytic switching. An emerging mechanism involved in regulating helix αC orientation is the allosteric coupling with kinase domain surfaces involved in homo- or heterodimerization. In this review, we discuss dimerization-mediated regulation of the rapidly accelerated fibrosarcoma (RAF) and eIF2α kinase families and draw parallels with the analogous behavior of the epidermal growth factor receptor (EGFR) and serine/threonine-protein kinase endoribonuclease 1 (IRE1)/ribonuclease L (RNAse L) kinase families. Given that resistance to RAF-targeted therapeutics often stems from dimerization-dependent mechanisms, we suggest that a better understanding of dimerization-induced allostery may assist in developing alternate therapeutic strategies.

An enhanced chemoenzymatic method for loading substrates onto carrier protein domains.

Non-ribosomal peptide synthetase (NRPS) machineries produce many medically relevant peptides that cannot be easily accessed by chemical synthesis. Thus, understanding NRPS mechanism is of crucial importance to allow efficient redesign of these machineries to produce new compounds. During NRPS-mediated synthesis, substrates are covalently attached to peptidyl carrier proteins (PCPs), and studies of NRPSs are impeded by difficulties in producing PCPs loaded with substrates. Different approaches to load substrates onto PCP domains have been described, but all suffer from difficulties in either the complexity of chemical synthesis or low enzymatic efficiency. Here, we describe an enhanced chemoenzymatic loading method that combines 2 approaches into a single, highly efficient one-pot loading reaction. First, d-pantetheine and ATP are converted into dephospho-coenzyme A via the actions of 2 enzymes from coenzyme A (CoA) biosynthesis. Next, phosphoadenylates are dephosphorylated using alkaline phosphatase to allow linker attachment to PCP domain by Sfp mutant R4-4, which is inhibited by phosphoadenylates. This route does not depend on activity of the commonly problematic dephospho-CoA kinase and, therefore, offers an improved method for substrate loading onto PCP domains.

How to polymerize in order to survive.

Perforation of membranes and pore formation is mediated by polymerization of proteins of the immune system, complement C9 and Perforin, which share the conserved MACPF domain. In this issue of Immunity, Baran et al. (2009) identify the molecular mechanism initiating polymerization as charge interactions in the MACPF domain.

A novel sensor to map auxin response and distribution at high spatio-temporal resolution.

Auxin is a key plant morphogenetic signal but tools to analyse dynamically its distribution and signalling during development are still limited. Auxin perception directly triggers the degradation of Aux/IAA repressor proteins. Here we describe a novel Aux/IAA-based auxin signalling sensor termed DII-VENUS that was engineered in the model plant Arabidopsis thaliana. The VENUS fast maturing form of yellow fluorescent protein was fused in-frame to the Aux/IAA auxin-interaction domain (termed domain II; DII) and expressed under a constitutive promoter. We initially show that DII-VENUS abundance is dependent on auxin, its TIR1/AFBs co-receptors and proteasome activities. Next, we demonstrate that DII-VENUS provides a map of relative auxin distribution at cellular resolution in different tissues. DII-VENUS is also rapidly degraded in response to auxin and we used it to visualize dynamic changes in cellular auxin distribution successfully during two developmental responses, the root gravitropic response and lateral organ production at the shoot apex. Our results illustrate the value of developing response input sensors such as DII-VENUS to provide high-resolution spatio-temporal information about hormone distribution and response during plant growth and development.

Kinetically distinct sorting pathways through the Golgi exhibit different requirements for Arf1.

To investigate the role of cytoplasmic sequences in directing transmembrane protein trafficking through the Golgi, we analyzed the sorting of VSV tsO45 G fusions with either the native G cytoplasmic domain (G) or an alternative cytoplasmic tail derived from the chicken AE1-4 anion exchanger (G(AE) ). At restrictive temperature G(AE) and G accumulated in the ER, and upon shifting the cells to permissive temperature both proteins folded and underwent transport through the Golgi. However, G(AE) and G did not form hetero-oligomers upon the shift to permissive temperature and they progressed through the Golgi with distinct kinetics. In addition, the transport of G through the proximal Golgi was Arf1 and COPI-dependent, while G(AE) progression through the proximal Golgi was Arf1 and COPI-independent. Although Arf1 did not regulate the sorting of G(AE) in the cis-Golgi, Arf1 did regulate the exit of G(AE) from the TGN. The trafficking of G(AE) through the Golgi was similar to that of the native AE1-4 anion exchanger, in that the progression of both proteins through the proximal Golgi was Arf1-independent, while both required Arf1 to exit the TGN. We propose that the differential recognition of cytosolic signals in membrane-spanning proteins by the Arf1-dependent sorting machinery may influence the rate at which cargo progresses through the Golgi.

Extension of Helix 12 in Munc18-1 Induces Vesicle Priming.

UNLABELLED: Munc18-1 is essential for vesicle fusion and participates in the docking of large dense-core vesicles to the plasma membrane. Recent structural data suggest that conformational changes in the 12th helix of the Munc18-1 domain 3a within the Munc18-1:syntaxin complex result in an additional interaction with synaptobrevin-2/VAMP2 (vesicle-associated membrane protein 2), leading to SNARE complex formation. To test this hypothesis in living cells, we examined secretion from Munc18-1-null mouse adrenal chromaffin cells expressing Munc18-1 mutants designed to either perturb the extension of helix 12 (Δ324-339), block its interaction with synaptobrevin-2 (L348R), or extend the helix to promote coil-coil interactions with other proteins (P335A). The mutants rescued vesicle docking and syntaxin-1 targeting to the plasma membrane, with the exception of P335A that only supported partial syntaxin-1 targeting. Disruptive mutations (L348R or Δ324-339) lowered the secretory amplitude by decreasing vesicle priming, whereas P335A markedly increased priming and secretory amplitude. The mutants displayed unchanged kinetics and Ca(2+) dependence of fusion, indicating that the mutations specifically affect the vesicle priming step. Mutation of a nearby tyrosine (Y337A), which interacts with closed syntaxin-1, mildly increased secretory amplitude. This correlated with results from an in vitro fusion assay probing the functions of Munc18-1, indicating an easier transition to the extended state in the mutant. Our findings support the notion that a conformational transition within the Munc18-1 domain 3a helix 12 leads to opening of a closed Munc18-1:syntaxin complex, followed by productive SNARE complex assembly and vesicle priming. SIGNIFICANCE STATEMENT: The essential postdocking role of Munc18-1 in vesicular exocytosis has remained elusive, but recent data led to the hypothesis that the extension of helix 12 in Munc18 within domain 3a leads to synaptobrevin-2/VAMP2 interaction and SNARE complex formation. Using both lack-of-function and gain-of-function mutants, we here report that the conformation of helix 12 predicts vesicle priming and secretory amplitude in living chromaffin cells. The effects of mutants on secretion could not be explained by differences in syntaxin-1 chaperoning/localization or vesicle docking, and the fusion kinetics and calcium dependence were unchanged, indicating that the effect of helix 12 extension is specific for the vesicle-priming step. We conclude that a conformational change within helix 12 is responsible for the essential postdocking role of Munc18-1 in neurosecretion.

SMP-domain proteins at membrane contact sites: Structure and function.

SMP-domains are found in proteins that localize to membrane contact sites. Elucidation of the properties of these proteins gives clues as to the molecular bases underlying processes that occur at such sites. Described here are recent discoveries concerning the structure, function, and regulation of the Extended-Synaptotagmin proteins and ERMES complex subunits, SMP-domain proteins at endoplasmic reticulum (ER)-plasma membrane and ER-mitochondrial contacts, respectively. They act as tethers contributing to the architecture of these sites and as lipid transporters that convey glycerolipids between apposed membranes. This article is part of a Special Issue entitled: The cellular lipid landscape edited by Tim P. Levine and Anant K. Menon.

A direct proofreader-clamp interaction stabilizes the Pol III replicase in the polymerization mode.

Processive DNA synthesis by the αεθ core of the Escherichia coli Pol III replicase requires it to be bound to the ß2 clamp via a site in the α polymerase subunit. How the ε proofreading exonuclease subunit influences DNA synthesis by α was not previously understood. In this work, bulk assays of DNA replication were used to uncover a non-proofreading activity of ε. Combination of mutagenesis with biophysical studies and single-molecule leading-strand replication assays traced this activity to a novel ß-binding site in ε that, in conjunction with the site in α, maintains a closed state of the αεθ-ß2 replicase in the polymerization mode of DNA synthesis. The ε-ß interaction, selected during evolution to be weak and thus suited for transient disruption to enable access of alternate polymerases and other clamp binding proteins, therefore makes an important contribution to the network of protein-protein interactions that finely tune stability of the replicase on the DNA template in its various conformational states.

ENTH and ANTH domain proteins participate in AP2-independent clathrin-mediated endocytosis.

Clathrin-mediated endocytosis (CME) is a major route of entry into eukaryotic cells. A core of evolutionarily ancient genes encodes many components of this system but much of our mechanistic understanding of CME is derived from a phylogenetically narrow sampling of a few model organisms. In the parasite Trypanosoma brucei, which is distantly related to the better characterised animals and fungi, exceptionally fast endocytic turnover aids its evasion of the host immune system. Although clathrin is absolutely essential for this process, the adaptor protein complex 2 (AP2) has been secondarily lost, suggesting mechanistic divergence. Here, we characterise two phosphoinositide-binding monomeric clathrin adaptors, T. brucei (Tb)EpsinR and TbCALM, which in trypanosomes are represented by single genes, unlike the expansions present in animals and fungi. Depletion of these gene products reveals essential, but partially redundant, activities in CME. Ultrastructural analysis of TbCALM and TbEpsinR double-knockdown cells demonstrated severe defects to clathrin-coated pit formation and morphology associated with a dramatic inhibition of endocytosis. Depletion of TbCALM alone, however, produced a distinct lysosomal segregation phenotype, indicating an additional non-redundant role for this protein. Therefore, TbEpsinR and TbCALM represent ancient phosphoinositide-binding proteins with distinct and vital roles in AP2-independent endocytosis.