Structure of the yeast Pml1 splicing factor and its integration into the RES complex

The RES complex was previously identified in yeast as a splicing factor affecting nuclear pre-mRNA retention. This complex was shown to contain three subunits, namely Snu17, Bud13 and Pml1, but its mode of action remains ill-defined. To obtain insights into its function, we have performed a structural investigation of this factor. Production of a short N-terminal truncation of residues that are apparently disordered allowed us to determine the X-ray crystallographic structure of Pml1. This demonstrated that it consists mainly of a FHA domain, a fold which has been shown to mediate interactions with phosphothreonine-containing peptides. Using a new sensitive assay based on alternative splice-site choice, we show, however, that mutation of the putative phosphothreonine-binding pocket of Pml1 does not affect pre-mRNA splicing. We have also investigated how Pml1 integrates into the RES complex. Production of recombinant complexes, combined with serial truncation and mutagenesis of their subunits, indicated that Pml1 binds to Snu17, which itself contacts Bud13. This analysis allowed us to demarcate the binding sites involved in the formation of this assembly. We propose a model of the organization of the RES complex based on these results, and discuss the functional consequences of this architecture.     

An unusual RNA recognition motif acts as a scaffold for multiple proteins in the pre-mRNA retention and splicing complex

The yeast pre-mRNA retention and splicing complex counteracts the escape of unspliced pre-mRNAs from the nucleus and activates splicing of a subset of Mer1p-dependent genes. A homologous complex is present in activated human spliceosomes. In many components of the spliceosome, RNA recognition motifs (RRMs) serve as versatile protein-RNA or protein-protein interaction platforms. Here, we show that in the retention and splicing complex, an atypical RRM of the Snu17p (small nuclear ribonucleoprotein-associated protein 17) subunit acts as a scaffold that organizes the other two constituents, Bud13p (bud site selection 13) and Pml1p (pre-mRNA leakage 1). GST pull-down experiments and size exclusion chromatography revealed that Snu17p constitutes the central platform of the complex, whereas Bud13p and Pml1p do not interact with each other. Fluorimetric structure probing showed the entire Bud13p and the N-terminal third of Pml1p to be natively disordered in isolation. Mutational analysis and tryptophan fluorescence confirmed that a conserved tryptophan-containing motif in the C terminus of Bud13p binds to the core RRM of Snu17p, whereas a different interaction surface encompassing a C-terminal extension of the Snu17p RRM is required to bind an N-terminal peptide of Pml1p. Isothermal titration calorimetry revealed 1:1 interaction stoichiometries, large negative binding entropies, and dissociation constants in the low nanomolar and micromolar ranges for the Snu17p-Bud13p and the Snu17p-Pml1p interactions, respectively. Our results demonstrate that the noncanonical Snu17p RRM concomitantly binds multiple ligand proteins via short, intrinsically unstructured peptide epitopes and thereby acts as a platform that displays functional modules of the ligands, such as a forkhead-associated domain of Pml1p and a conserved polylysine motif of Bud13p.     

Proteomic analysis identifies a new complex required for nuclear pre-mRNA retention and splicing

Using the proteomic tandem affinity purification (TAP) method, we have purified the Saccharomyces cerevisie U2 snRNP-associated splicing factors SF3a and SF3b. While SF3a purification revealed only the expected subunits Prp9p, Prp11p and Prp21p, yeast SF3b was found to contain only six subunits, including previously known components (Rse1p, Hsh155p, Cus1p, Hsh49p), the recently identified Rds3p factor and a new small essential protein (Ysf3p) encoded by an unpredicted split ORF in the yeast genome. Surprisingly, Snu17p, the proposed yeast orthologue of the seventh human SF3b subunit, p14, was not found in the yeast complex. TAP purification revealed that Snu17p, together with Bud13p and a newly identified factor, Pml1p/Ylr016c, form a novel trimeric complex. Subunits of this complex were not essential for viability. However, they are required for efficient splicing in vitro and in vivo. Furthermore, inactivation of this complex causes pre-mRNA leakage from the nucleus. The corresponding complex was named pre-mRNA REtention and Splicing (RES). The presence of RES subunit homologues in numerous eukaryotes suggests that its function is evolutionarily conserved.     

Molecular interactions of the Saccharomyces cerevisiae Atg1 complex provide insights into assembly and regulatory mechanisms

The Atg1 complex, which contains 5 major subunits: Atg1, Atg13, Atg17, Atg29, and Atg31, regulates the induction of autophagy and autophagosome formation. To gain a better understanding of the overall architecture and assembly mechanism of this essential autophagy regulatory complex, we have reconstituted a core assembly of the Saccharomyces cerevisiae Atg1 complex composed of full-length Atg17, Atg29, and Atg31, along with the C-terminal domains of Atg1 (Atg1[CTD]) and Atg13 (Atg13[CTD]). Using chemical-crosslinking coupled with mass spectrometry (CXMS) analysis we systematically mapped the intersubunit interaction interfaces within this complex. Our data revealed that the intrinsically unstructured C-terminal domain of Atg29 interacts directly with Atg17, whereas Atg17 interacts with Atg13 in 2 distinct intrinsically unstructured regions, including a previously unknown motif that encompasses several putative phosphorylation sites. The Atg1[CTD] crosslinks exclusively to the Atg13[CTD] and does not appear to make direct contact with the Atg17-Atg31-Atg29 scaffold. Finally, single-particle electron microscopy analysis revealed that both the Atg13[CTD] and Atg1[CTD] localize to the tip regions of Atg17-Atg31-Atg29 and do not alter the distinct curvature of this scaffolding subcomplex. This work provides a comprehensive understanding of the subunit interactions in the fully assembled Atg1 core complex, and uncovers the potential role of intrinsically disordered regions in regulating complex integrity.     

Structural and Functional Studies of the Yeast Class II Hda1 Histone Deacetylase Complex

Yeast class II Hda1 histone deacetylase (HDAC) complex is an H2B- and H3-specific HDAC in Saccharomyces cerevisiae consisting of three previously identified subunits, the catalytic subunit scHda1p and two non-catalytic structural subunits scHda2p and scHda3p. We co-expressed and co-purified recombinant yeast class II HDAC complex from bacteria as a functionally active and trichostatin-A-sensitive hetero-tetrameric complex. According to an extensive analysis of domain organization and interaction of all subunits (or domains), the N-terminal domain of scHda1p associates through the C-terminal coiled-coil domains (CCDs) of the scHda2p-scHda3p sub-complex, yielding a truncated scHda1pHDAC-scHda2pCCD2-scHda3pCCD3 complex with indistinguishable deacetylase activity compared to the full-length complex in vitro. We characterized the interaction of the HDAC complex with either single-stranded or double-stranded DNA and identified the N-terminal halves of scHda2p and scHda3p as binding modules. A high-resolution structure of the scHda3p DNA-binding domain by X-ray crystallography is presented. The crystal structure shows an unanticipated structural homology with the C-terminal helicase lobes of SWI2/SNF2 chromatin-remodeling domains of the Rad54 family enzymes. DNA binding is unspecific for nucleotide sequence and structure, similar to the Rad54 enzymes in vitro. Our structural and functional analyses of the budding yeast class II Hda1 HDAC complex provide insight into DNA recognition and deacetylation of histones in nucleosomes.     

Chemical shift assignments of Mb1858 (24-155), a FHA domain-containing protein from <Emphasis Type="Italic">Mycobacterium bovis</Emphasis>

The forkhead-associated (FHA) domain is known as a phosphopeptide recognition domain embedded in regulatory proteins from both eukaryotes and bacteria with various biological functions. In this study, the gene encoding a predicted FHA domain from Mb1858 (residues V24-D155 from the 162 amino acid protein Mb1858) in Mycobacterium bovis was cloned, and U-¹³C/¹⁵N-labeled protein was prepared for backbone and side chain resonance assignments by NMR spectroscopy. These assignments are preliminary work towards the determination of the structure and phosphopeptide-binding properties using NMR methods, which will provide useful information about the function of Mb1858 protein.     

Direct interaction of DNA repair protein tyrosyl DNA phosphodiesterase 1 and the DNA ligase III catalytic domain is regulated by phosphorylation of its flexible N-terminus

Tyrosyl DNA phosphodiesterase 1 (TDP1) and DNA Ligase IIIα (LigIIIα) are key enzymes in single-strand break (SSB) repair. TDP1 removes 3′-tyrosine residues remaining after degradation of DNA topoisomerase (TOP) 1 cleavage complexes trapped by either DNA lesions or TOP1 inhibitors. It is not known how TDP1 is linked to subsequent processing and LigIIIα-catalyzed joining of the SSB. Here we define a direct interaction between the TDP1 catalytic domain and the LigIII DNA-binding domain (DBD) regulated by conformational changes in the unstructured TDP1 N-terminal region induced by phosphorylation and/or alterations in amino acid sequence. Full-length and N-terminally truncated TDP1 are more effective at correcting SSB repair defects in TDP1 null cells compared with full-length TDP1 with amino acid substitutions of an N-terminal serine residue phosphorylated in response to DNA damage. TDP1 forms a stable complex with LigIII_170–755, as well as full-length LigIIIα alone or in complex with the DNA repair scaffold protein XRCC1. Small-angle X-ray scattering and negative stain electron microscopy combined with mapping of the interacting regions identified a TDP1/LigIIIα compact dimer of heterodimers in which the two LigIII catalytic cores are positioned in the center, whereas the two TDP1 molecules are located at the edges of the core complex flanked by highly flexible regions that can interact with other repair proteins and SSBs. As TDP1and LigIIIα together repair adducts caused by TOP1 cancer chemotherapy inhibitors, the defined interaction architecture and regulation of this enzyme complex provide insights into a key repair pathway in nonmalignant and cancer cells.     

The FHA domain determines Drosophila Chk2/Mnk localization to key mitotic structures and is essential for early embryonic DNA damage responses

DNA damage responses, including mitotic centrosome inactivation, cell-cycle delay in mitosis, and nuclear dropping from embryo cortex, maintain genome integrity in syncytial Drosophila embryos. A conserved signaling kinase, Chk2, known as Mnk/Loki, is essential for the responses. Here we demonstrate that functional EGFP-Mnk expressed from a transgene localizes to the nucleus, centrosomes, interkinetochore/centromere region, midbody, and pseudocleavage furrows without DNA damage and in addition forms numerous foci/aggregates on mitotic chromosomes upon DNA damage. We expressed EGFP-tagged Mnk deletion or point mutation variants and investigated domain functions of Mnk in vivo. A triple mutation in the phosphopeptide-binding site of the forkhead-associated (FHA) domain disrupted normal Mnk localization except to the nucleus. The mutation also disrupted Mnk foci formation on chromosomes upon DNA damage. FHA mutations and deletion of the SQ/TQ-cluster domain (SCD) abolished Mnk transphosphorylations and autophosphorylations, indicative of kinase activation after DNA damage. A potent NLS was found at the C-terminus, which is required for normal Mnk function. We propose that the FHA domain in Mnk plays essential dual functions in mediating embryonic DNA damage responses by means of its phosphopeptide-binding ability: activating Mnk in the nucleus upon DNA damage and recruiting Mnk to multiple subcellular structures independently of DNA damage.     

Spa2p Interacts with Cell Polarity Proteins and Signaling Components Involved in Yeast Cell Morphogenesis

The yeast protein Spa2p localizes to growth sites and is important for polarized morphogenesis during budding, mating, and pseudohyphal growth. To better understand the role of Spa2p in polarized growth, we analyzed regions of the protein important for its function and proteins that interact with Spa2p. Spa2p interacts with Pea2p and Bud6p (Aip3p) as determined by the two-hybrid system; all of these proteins exhibit similar localization patterns, and spa2Δ, pea2Δ, and bud6Δ mutants display similar phenotypes, suggesting that these three proteins are involved in the same biological processes. Coimmunoprecipitation experiments demonstrate that Spa2p and Pea2p are tightly associated with each other in vivo. Velocity sedimentation experiments suggest that a significant portion of Spa2p, Pea2p, and Bud6p cosediment, raising the possibility that these proteins form a large, 12S multiprotein complex. Bud6p has been shown previously to interact with actin, suggesting that the 12S complex functions to regulate the actin cytoskeleton. Deletion analysis revealed that multiple regions of Spa2p are involved in its localization to growth sites. One of the regions involved in Spa2p stability and localization interacts with Pea2p; this region contains a conserved domain, SHD-II. Although a portion of Spa2p is sufficient for localization of itself and Pea2p to growth sites, only the full-length protein is capable of complementing spa2 mutant defects, suggesting that other regions are required for Spa2p function. By using the two-hybrid system, Spa2p and Bud6p were also found to interact with components of two mitogen-activated protein kinase (MAPK) pathways important for polarized cell growth. Spa2p interacts with Ste11p (MAPK kinase [MEK] kinase) and Ste7p (MEK) of the mating signaling pathway as well as with the MEKs Mkk1p and Mkk2p of the Slt2p (Mpk1p) MAPK pathway; for both Mkk1p and Ste7p, the Spa2p-interacting region was mapped to the N-terminal putative regulatory domain. Bud6p interacts with Ste11p. The MEK-interacting region of Spa2p corresponds to the highly conserved SHD-I domain, which is shown to be important for mating and MAPK signaling. spa2 mutants exhibit reduced levels of pheromone signaling and an elevated level of Slt2p kinase activity. We thus propose that Spa2p, Pea2p, and Bud6p function together, perhaps as a complex, to promote polarized morphogenesis through regulation of the actin cytoskeleton and signaling pathways.     

A calcium-driven conformational switch of the N-terminal and core domains of annexin A1

In 1993, Huber and co-workers published the structure of an N-terminally truncated version of human annexin A1 lacking the first 32 amino acid residues (PDB code: 1AIN). In 2001, we reported the structure of full-length porcine annexin A1 including the N-terminal domain in the absence of calcium ions (PDB code: 1HM6). The latter structure did not reflect a typical annexin core fold, but rather a surprising interaction of the N-terminal domain and the core domain. Comparing these two structures revealed that in the full-length structure the first 12 residues of the N-terminal domain insert into the core of the protein, thereby replacing and unwinding one of the alpha-helices (helix D in repeat 3) that is involved in calcium binding. We hypothesized that this structure in the absence of calcium ions represents the inactive form of the protein. Furthermore, we proposed that upon calcium binding, the N-terminal domain would be expelled from the core domain and that the core D-helix would reform in the proper conformation for calcium coordination. Herein, we report the X-ray structure of full-length porcine annexin A1 in the presence of calcium. This new structure shows a typical annexin core structure as we hypothesized, with the D-helix back in place for calcium coordination while parts of the now exposed N-terminal domain are disordered. We could locate eight calcium ions in this structure, two of which are octa-coordinated and two of which were not observed in the structure of the N-terminally truncated annexin A1. Possible implications of this calcium-induced conformational switch for the membrane aggregation properties of annexin A1 will be discussed.     

A hybrid structural model of the complete Brugia malayi cytoplasmic asparaginyl-tRNA synthetase

Aminoacyl-tRNA synthetases are validated molecular targets for anti-infective drug discovery because of their essentiality in protein synthesis. Thanks to genome sequencing, it is now possible to systematically study aminoacyl-tRNA synthetases from human eukaryotic parasites as putative targets for novel drug discovery. As part of a program targeting class IIb asparaginyl-tRNA synthetases (AsnRS) from the parasitic nematode Brugia malayi for anti-filarial drugs, we report the complete structure of a eukaryotic AsnRS. Metazoan and fungal AsnRS differ from their bacterial homologues by the addition of a conserved N-terminal extension of about 110 residues whose structure we have determined by solution NMR for the B. malayi enzyme. In addition, we solved by X-ray crystallography a series of structures of the catalytically active N-terminally truncated enzyme (residues 112-548), allowing the structural basis for the mechanism of asparagine activation to be elucidated. The N-terminal domain contains a structured region with a novel fold featuring a lysine-rich helix that is shown by NMR to interact with tRNA. This is connected by an unstructured tether to the remainder of the enzyme, which is highly similar to the known structure of bacterial AsnRS. These data enable a model of the complete AsnRS-tRNA complex to be constructed.     

Distinct roles of the N-terminal and C-terminal precursor domains in the biogenesis of the Bordetella pertussis filamentous hemagglutinin.

The 220-kDa Bordetella pertussis filamentous hemagglutinin (FHA) is the major exported protein found in culture supernatants. The structural gene of FHA has a coding potential for a 367-kDa protein, and the mature form constitutes the N-terminal 60% of the 367-kDa precursor. The C-terminal domain of the precursor was found to be important for the high-level secretion of full-length FHA but not of truncated analogs (80 kDa or less). The secretion of full-length and truncated FHA polypeptides requires the presence of the approximately 100-amino-acid N-terminal domain and the outer membrane protein FhaC, homologous to the N-terminal domains of the Serratia marcescens and Proteus mirabilis hemolysins and their accessory proteins, respectively. By analogy to these hemolysins, it is likely that the N-terminal domain of the FHA precursor interacts, directly or indirectly, with the accessory protein during FHA biogenesis. However, immunogenicity and antigenicity studies suggest that the N-terminal domain of FHA is masked by its C-terminal domain and therefore should not be available for its interactions with FhaC. These observations suggest a model in which the C-terminal domain of the FHA precursor may play a role as an intramolecular chaperone to prevent premature folding of the protein. Both heparin binding and hemagglutination are expressed by the N-terminal half of FHA, indicating that this domain contains important functional regions of the molecule.     

The FHA domain is a modular phosphopeptide recognition motif.

FHA domains are conserved sequences of 65-100 amino acid residues found principally within eukaryotic nuclear proteins, but which also exist in certain prokaryotes. The FHA domain is thought to mediate protein-protein interactions, but its mode of action has yet to be elucidated. Here, we show that the two highly divergent FHA domains of Saccharomyces cerevisiae Rad53p, a protein kinase involved in cell cycle checkpoint control, possess phosphopeptide-binding specificity. We also demonstrate that other FHA domains bind peptides in a phospho-dependent manner. These findings indicate that the FHA domain is a phospho-specific protein-protein interaction motif and have important implications for mechanisms of intracellular signaling in both eukaryotes and prokaryotes.     

The FHA domain of OdhI interacts with the carboxyterminal 2-oxoglutarate dehydrogenase domain of OdhA in Corynebacterium glutamicum

In Corynebacterium glutamicum, the unphosphorylated 15-kDa OdhI protein inhibits the activity of the 2-oxoglutarate dehydrogenase complex (ODHc) by binding to OdhA, which in corynebacteria and mycobacteria is a large fusion protein with two major domains exhibiting structural features of E1o and E2 proteins. Using copurification and surface plasmon resonance experiments with different OdhI and OdhA length variants it was shown that the entire forkhead-associated (FHA) domain of OdhI and the C-terminal dehydrogenase domain of OdhA are required for interaction. The FHA domain was also sufficient for inhibition of ODHc activity. Phosphorylated OdhI was binding-incompetent and did not inhibit ODHc activity.

Structured summary

MINT-7713362:OdhI (uniprotkb:Q8NQJ3) binds (MI:0407) to OdhA (uniprotkb:Q8NRC3) by surface plasmon resonance (MI:0107)MINT-7713261:OdhI (uniprotkb:Q8NQJ3) physically interacts (MI:0915) with OdhA (uniprotkb:Q8NRC3) by pull down (MI:0096)     

Subcellular localization of the interaction of bipolar landmarks Bud8p and Bud9p with Rax2p in Saccharomyces cerevisiae diploid cells

In Saccharomyces cerevisiae, the bud site selection of diploid cells is regulated by at least four persistent landmarks, Bud8p, Bud9p, Rax1p, and Rax2p. Bud8p and Bud9p are essential for the establishment of bipolar budding and localize mainly to the distal and the proximal poles, respectively. Their subcellular localizations are regulated through interaction with Rax1p/Rax2p. We investigated when and where Bud8p and Bud9p physically interact with Rax2p in vivo using a split-GFP method. GFP fluorescence showed that Bud8p physically interacted with Rax2p at the proximal or distal pole in unbudded cells; a physical interaction was also observed at the opposite pole to the growing bud in mother cells with a large-size bud. Bud9p physically interacted with Rax2p at the birth scar in budded mother cells. These observations suggest that the interaction of Rax2p with Bud8p and Bud9p may contribute to the translocation of bipolar landmarks to the correct sites.     

Two subclasses of guanine exchange factor (GEF) domains revealed by comparison of activities of chimeric genes constructed from CDC25, SDC25 and BUD5 in Saccharomyces cerevisiae

Guanine Exchange Factor (GEF) activity for Ras proteins has been associated with a conserved domain in Cdc25p, Sdc25p in Saccharomyces cerevisiae and several other proteins recently found in other eukaryotes. We have assessed the structure-function relationships between three different members of this family in S. cerevisiae, Cdc25p, Sdc25p and Bud5p. Cdc25p controls the Ras pathway, whereas Bud5p controls bud site localization. We demonstrate that the GEF domain of Sdc25p is closely related to that of Cdc25p. We first constructed a thermosensitive allele of SDC25 by specifically altering amino acid positions known to be changed in the cdc25-1 mutation. Secondly, we constructed three chimeric genes from CDC25 and SDC25, the products of which are as active in the Ras pathway as are the wild-type proteins. In contrast, similar chimeras made between CDC25 and BUD5 lead to proteins that are inactive both in the Ras and budding control pathways. This difference in the ability of chimeric proteins to retain activity allows us to define two subclasses of structurally different GEFs: Cdc25p and Sdc25p are Ras-specific GEFs, and Bud5p is a putative GEF for the Rsr1/Bud1 Rap-like protein.     

Crystal structures of the response regulator DosR from Mycobacterium tuberculosis suggest a helix rearrangement mechanism for phosphorylation activation

The response regulator DosR is essential for promoting long-term survival of Mycobacterium tuberculosis under low oxygen conditions in a dormant state and may be responsible for latent tuberculosis in one-third of the world's population. Here, we report crystal structures of full-length unphosphorylated DosR at 2.2 Å resolution and its C-terminal DNA-binding domain at 1.7 Å resolution. The full-length DosR structure reveals several features never seen before in other response regulators. The N-terminal domain of the full-length DosR structure has an unexpected (βα)₄ topology instead of the canonical (βα)₅ fold observed in other response regulators. The linker region adopts a unique conformation that contains two helices forming a four-helix bundle with two helices from another subunit, resulting in dimer formation. The C-terminal domain in the full-length DosR structure displays a novel location of helix α10, which allows Gln199 to interact with the catalytic Asp54 residue of the N-terminal domain. In contrast, the structure of the DosR C-terminal domain alone displays a remarkable unstructured conformation for helix α10 residues, different from the well-defined helical conformations in all other known structures, indicating considerable flexibility within the C-terminal domain. Our structures suggest a mode of DosR activation by phosphorylation via a helix rearrangement mechanism.     

Identification of the Interactors of Human Nibrin (NBN) and of Its 26 kDa and 70 kDa Fragments Arising from the NBN 657del5 Founder Mutation

Nibrin (also named NBN or NBS1) is a component of the MRE11/RAD50/NBN complex, which is involved in early steps of DNA double strand breaks sensing and repair. Mutations within the NBN gene are responsible for the Nijmegen breakage syndrome (NBS). The 90% of NBS patients are homozygous for the 657del5 mutation, which determines the synthesis of two truncated proteins of 26 kDa (p26) and 70 kDa (p70). Here, HEK293 cells have been exploited to transiently express either the full-length NBN protein or the p26 or p70 fragments, followed by affinity chromatography enrichment of the eluates. The application of an unsupervised proteomics approach, based upon SDS-PAGE separation and shotgun digestion of protein bands followed by MS/MS protein identification, indicates the occurrence of previously unreported protein interacting partners of the full-length NBN protein and the p26 fragment containing the FHA/BRCT1 domains, especially after cell irradiation. In particular, results obtained shed light on new possible roles of NBN and of the p26 fragment in ROS scavenging, in the DNA damage response, and in protein folding and degradation. In particular, here we show that p26 interacts with PARP1 after irradiation, and this interaction exerts an inhibitory effect on PARP1 activity as measured by NAD⁺ levels. Furthermore, the p26-PARP1 interaction seems to be responsible for the persistence of ROS, and in turn of DSBs, at 24 h from IR. Since some of the newly identified interactors of the p26 and p70 fragments have not been found to interact with the full-length NBN, these interactions may somehow contribute to the key biological phenomena underpinning NBS.