Homodimerization Domain Research Articles

In-depth Characterization of the Homodimerization Domain of the Transcription Factor THAP1 and Dystonia-Causing Mutations Therein.

Mutations in the THAP1 gene encoding the transcription factor THAP1 have been shown to cause DYT6 dystonia. THAP1 contains a highly conserved THAP zinc finger at its N-terminal region which allows specific binding to its target sequences as well as a coiled-coil domain (amino acids 139-190) towards its C-terminus postulated as a protein-protein-binding motif. While several DYT6-causing mutations within the THAP domain were shown to decrease THAP1 activity in transcriptional regulation and DNA-binding, the role of mutations within the coiled-coil domain is rather unknown. Therefore, assigning a function to this domain may enable functional testing of mutations in this region. Notably, THAP1 and other THAP proteins form homodimers; however, the responsible domain has not been elucidated in detail. We show that the region of amino acids 139-185 is involved in formation of THAP1 homodimers by using yeast-two-hybrid, GST pull-down, and cross-linking assays. Surprisingly, all nine reported DYT6-causing missense mutations within this region had no effect on dimerization of THAP1 in GST pull-down and formaldehyde cross-linking assays. In conclusion, we demonstrated that a region of 47 amino acids is involved in THAP1 homodimerization but mutations in this region seem not to impair this mechanism.

Structure of the homodimeric androgen receptor ligand-binding domain

The androgen receptor (AR) plays a crucial role in normal physiology, development and metabolism as well as in the aetiology and treatment of diverse pathologies such as androgen insensitivity syndromes (AIS), male infertility and prostate cancer (PCa). Here we show that dimerization of AR ligand-binding domain (LBD) is induced by receptor agonists but not by antagonists. The 2.15-Å crystal structure of homodimeric, agonist- and coactivator peptide-bound AR-LBD unveils a 1,000-Å2 large dimerization surface, which harbours over 40 previously unexplained AIS- and PCa-associated point mutations. An AIS mutation in the self-association interface (P767A) disrupts dimer formation in vivo, and has a detrimental effect on the transactivating properties of full-length AR, despite retained hormone-binding capacity. The conservation of essential residues suggests that the unveiled dimerization mechanism might be shared by other nuclear receptors. Our work defines AR-LBD homodimerization as an essential step in the proper functioning of this important transcription factor.

A Survey of Fusion Genes in Myeloma Identifies Kinase Domain Activation Which Could be Targeted with Available Treatments

IntroductionAlthough fusion genes other than the immunoglobulin (Ig) translocation, t(4;14), which results in IGH-WHSC1fusions, are not frequently detected in multiple myeloma (MM), recent evidence suggests that kinase fusion gene fusions do occur relatively frequently and may inform on treatment algorithms. Here we use a hybrid-capture based, next-generation sequencing assay to survey fusion genes in patients with MM.MethodsWe report on 1421 samples from 958 individuals diagnosed with either monoclonal gammopathy of unknown significance (MGUS), smoldering multiple myeloma (SMM) or MM who underwent targeted sequencing with the FoundationOne Heme® (F1H) assay. Tumor samples were obtained from bone marrow aspirates, enriched by CD138+selection using magnetic beads (AutoMACs, Miltenyi Biotech, Cologne, Germany or RoboSep, StemCell Technologies, Vancouver, Canada). RNA and DNA were extracted using the AllPrep DNA/RNA mini kit (Qiagen, Hilden, Germany), RNeasy RNA extraction kit (Qiagen) or Puregene DNA extraction kit (Qiagen). ≥ 50 ng of extracted DNA or RNA was processed on the F1H assay. The current assay analyzes the complete coding DNA sequence of 405 genes, as well as selected introns of 31 genes involved in chromosomal rearrangements as well as the RNA sequence of 265 commonly rearranged genes resulting in gene fusions. Genes included in this assay encode known or likely targets of therapy, either approved or in clinical trials, or are otherwise known drivers of oncogenesis. Sequencing was to an average depth of 510x and was performed using the Illumina HiSeq 2500. Sequences were analyzed for selected gene rearrangements including fusion genes which were detected by a combination of DNA and RNA sequencing.ResultsRearrangements into the Ig loci were detected and included the 5 main translocations: t(4;14), t(6;14), t(11;14), t(14;16), and t(14;20), as well as translocations involving MYC at 8q24. From a combination of DNA capture and RNA-seq expression values we used 107 samples in a training set with matching gene expression profiling data to determine cut-offs for FGFR3, WHSC1, CCND3, MAF, MAFB, CCND2 and CCND1to stratify patients into the 5 main translocation groups. We used these values to classify a further 391 samples with corresponding gene expression profiling (GEP) data, resulting in sensitivities and specificities of t(4;14), 98% and 100%; t(6;14), 100% and 99%; t(11;14), 99% and 95%; t(14;16), 77% and 100%; t(14;20), 100% and 100%, respectively.40 non-Ig rearrangements were detected in 38 patients (4.2%), of which 21 in-frame fusion genes were predicted. Recurrent fusion-genes, identified in more than one patient, included EIF4E3-FOXP1, TXNDC5-MYC and SUB1-WHSC1. As well as TXNDC5, MYC was also partnered with FOXO3, both of which are known partners of the MYC translocation. 12 of the 21 in-frame fusion genes involved kinase domains, including fusions with BRAF (n=4), NTRK3 (n=2), ALK (n=1), ROS1 (n=1), MAPK14 (n=1), MAP3K14 (n=1), FGFR1 (n=1), and DLG2 (n=1). Fusions involving each of these genes have been documented in other cancers. BRAF fusions are thought to partner with genes encoding homodimerization domains, resulting in downstream activation of Ras signaling. Other kinase fusions result in receptor signaling and downstream activation of the Ras signaling pathway. Of the patients with kinase fusions, 2 had an activating KRAS, NRAS or BRAF mutation but only one was clonal (84% cancer clonal fraction). One patient with samples taken at different timepoints had a GTF2I-BRAF fusion and concomitant KRAS G13C mutation (16% allele frequency), both of which were not detectable 8 months later but an AGK-BRAF fusion was detected at that time suggesting clonal selection.ConclusionNon-Ig fusion genes are present in myeloma patients, but at a low frequency. Most of the fusions detected contained a kinase domain indicating activation of the Ras signaling pathway, which is also activated through KRAS, NRAS and BRAF mutations in 50% of patients. Although rare (1%), these kinase fusions are potential clinical targets in myeloma where kinase inhibitors, such as crizotinib, can be used which has shown to be effective against ALK and ROS1 fusions. DisclosuresHe:Foundation Medicine, Inc: Employment, Equity Ownership. Zhong:foundation medicine: Employment. Bailey:Foundation Medicine, Inc: Employment, Equity Ownership. Vergillo:Foundation Medicine, Inc: Employment. Ross:Foundation Medicine, Inc: Employment. Miller:Foundation Medicine: Employment, Equity Ownership. Stephens:Foundation Medicine: Employment, Equity Ownership. Mughal:Foundation Medicine: Employment, Equity Ownership. Davies:Celgene: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Morgan:Janssen: Research Funding; Univ of AR for Medical Sciences: Employment; Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Bristol Meyers: Consultancy, Honoraria.

Characterization of Atg38 and NRBF2, a fifth subunit of the autophagic Vps34/PIK3C3 complex

ABSTRACTThe phosphatidylinositol 3-kinase Vps34 is part of several protein complexes. The structural organization of heterotetrameric complexes is starting to emerge, but little is known about organization of additional accessory subunits that interact with these assemblies. Combining hydrogen-deuterium exchange mass spectrometry (HDX-MS), X-ray crystallography and electron microscopy (EM), we have characterized Atg38 and its human ortholog NRBF2, accessory components of complex I consisting of Vps15-Vps34-Vps30/Atg6-Atg14 (yeast) and PIK3R4/VPS15-PIK3C3/VPS34-BECN1/Beclin 1-ATG14 (human). HDX-MS shows that Atg38 binds the Vps30-Atg14 subcomplex of complex I, using mainly its N-terminal MIT domain and bridges the coiled-coil I regions of Atg14 and Vps30 in the base of complex I. The Atg38 C-terminal domain is important for localization to the phagophore assembly site (PAS) and homodimerization. Our 2.2 Å resolution crystal structure of the Atg38 C-terminal homodimerization domain shows 2 segments of α-helices assembling into a mushroom-like asymmetric homodimer with a 4-helix cap and a parallel coiled-coil stalk. One Atg38 homodimer engages a single complex I. This is in sharp contrast to human NRBF2, which also forms a homodimer, but this homodimer can bridge 2 complex I assemblies.

Three-dimensional structure of homodimeric androgen receptor ligand-binding domain

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

PDE11A negatively regulates lithium responsivity

Lithium responsivity in patients with bipolar disorder has been genetically associated with Phosphodiesterase 11A (PDE11A), and lithium decreases PDE11A mRNA in induced pluripotent stem cell-derived hippocampal neurons originating from lithium-responsive patients. PDE11 is an enzyme uniquely enriched in the hippocampus that breaks down cyclic AMP and cyclic GMP. Here we determined whether decreasing PDE11A expression is sufficient to increase lithium responsivity in mice. In dorsal hippocampus and ventral hippocampus (VHIPP), lithium-responsive C57BL/6J and 129S6/SvEvTac mice show decreased PDE11A4 protein expression relative to lithium-unresponsive BALB/cJ mice. In VHIPP, C57BL/6J mice also show differences in PDE11A4 compartmentalization relative to BALB/cJ mice. In contrast, neither PDE2A nor PDE10A expression differ among the strains. The compartment-specific differences in PDE11A4 protein expression are explained by a coding single-nucleotide polymorphism (SNP) at amino acid 499, which falls within the GAF-B homodimerization domain. Relative to the BALB/cJ 499T, the C57BL/6J 499A decreases PDE11A4 homodimerization, which removes PDE11A4 from the membrane. Consistent with the observation that lower PDE11A4 expression correlates with better lithium responsiveness, we found that Pde11a knockout mice (KO) given 0.4% lithium chow for 3+ weeks exhibit greater lithium responsivity relative to wild-type (WT) littermates in tail suspension, an antidepressant-predictive assay, and amphetamine hyperlocomotion, an anti-manic predictive assay. Reduced PDE11A4 expression may represent a lithium-sensitive pathophysiology, because both C57BL/6J and Pde11a KO mice show increased expression of the pro-inflammatory cytokine interleukin-6 (IL-6) relative to BALB/cJ and PDE11A WT mice, respectively. Our finding that PDE11A4 negatively regulates lithium responsivity in mice suggests that the PDE11A SNPs identified in patients may be functionally relevant.

The epigenetic regulator Smchd1 contains a functional GHKL-type ATPase domain.

Structural maintenance of chromosomes flexible hinge domain containing 1 (Smchd1) is an epigenetic regulator that plays critical roles in gene regulation during development. Mutations in SMCHD1 were recently implicated in the pathogenesis of facioscapulohumeral muscular dystrophy (FSHD), although the mechanistic basis remains of outstanding interest. We have previously shown that Smchd1 associates with chromatin via its homodimeric C-terminal hinge domain, yet little is known about the function of the putative GHKL (gyrase, Hsp90, histidine kinase, MutL)-type ATPase domain at its N-terminus. To formally assess the structure and function of Smchd1's ATPase domain, we have generated recombinant proteins encompassing the predicted ATPase domain and the adjacent region. Here, we show that the Smchd1 N-terminal region exists as a monomer and adopts a conformation resembling that of monomeric full-length heat shock protein 90 (Hsp90) protein in solution, even though the two proteins share only ∼8% overall sequence identity. Despite being monomeric, the N-terminal region of Smchd1 exhibits ATPase activity, which can be antagonized by the reaction product, ADP, or the Hsp90 inhibitor, radicicol, at a nanomolar concentration. Interestingly, introduction of an analogous mutation to that identified in SMCHD1 of an FSHD patient compromised protein stability, suggesting a possible molecular basis for loss of protein function and pathogenesis. Together, these results reveal important structure-function characteristics of Smchd1 that may underpin its mechanistic action at the chromatin level.

A Phytochrome Sensory Domain Permits Receptor Activation by Red Light.

Optogenetics and photopharmacology enable the spatio-temporal control of cell and animal behavior by light. Although red light offers deep-tissue penetration and minimal phototoxicity, very few red-light-sensitive optogenetic methods are currently available. We have now developed a red-light-induced homodimerization domain. We first showed that an optimized sensory domain of the cyanobacterial phytochrome 1 can be expressed robustly and without cytotoxicity in human cells. We then applied this domain to induce the dimerization of two receptor tyrosine kinases-the fibroblast growth factor receptor 1 and the neurotrophin receptor trkB. This new optogenetic method was then used to activate the MAPK/ERK pathway non-invasively in mammalian tissue and in multicolor cell-signaling experiments. The light-controlled dimerizer and red-light-activated receptor tyrosine kinases will prove useful to regulate a variety of cellular processes with light.

Mechanisms of Bacterial ABC Importers: Lessons from Structural and Functional Studies of the Ribose Transporter

Bacterial ATP-Binding Cassette (ABC) transporters are vital for the uptake of nutrients, including sugars, amino acids, and trace metals. Recent studies have identified two types of importers (Class I and II) based on mechanistic and structural differences. The ribose transporter, RbsABC, is a tripartite ABC importer consisting of a cytoplasmic ABC protein with fused nucleotide-binding domains (NBD), a transmembrane domain (TMD) homodimer, RbsC, and a periplasmic substrate binding protein (SBP), RbsB. However, RbsABC is divergent from canonical importers in the fused ATP-binding cassette, which contains both a consensus and degenerate NBD. This organization is observed in eukaryotic ABC exporters, but is thus far unique among characterized bacterial importers. Despite the asymmetry in the NBDs, significant conformational changes are observed in RbsB during transport based on distance measurements from EPR spectroscopy experiments. These changes are driven by the hydrolysis of ATP, providing a pathway for ribose release from RbsB and subsequent transfer to the cytoplasm. Biochemical and EPR experiments also demonstrate that the association between RbsA and RbsC is sensitive to the availability of nucleotide and stimulates ATP hydrolysis in RbsA, providing tight regulation of the catalytic cycle. Finally, it appears a single consensus ATPase site provides ATP hydrolysis to fuel transport, with rescue mutations failing to restore full activity in the degenerate site. EPR experiments suggest an asymmetric closure of the NBDs in response to magnesium and ATP availability. Whether this proposed opening and closing at a single NBD is sufficient to drive transport of a single ribose molecule is currently unknown. The combined observations suggest RbsABC functions by a mechanism distinct from the well-characterized ABC importers for maltose and vitamin B12. Furthermore, RbsABC may provide a key evolutionary link between bacterial and eukaryotic transporters.

Arm-in-Arm Response Regulator Dimers Promote Intermolecular Signal Transduction.

Bacteriophytochrome photoreceptors (BphPs) and their cognate response regulators make up two-component signal transduction systems which direct bacteria to mount phenotypic responses to changes in environmental light quality. Most of these systems utilize single-domain response regulators to transduce signals through unknown pathways and mechanisms. Here we describe the photocycle and autophosphorylation kinetics of RtBphP1, a red light-regulated histidine kinase from the desert bacterium Ramlibacter tataouinensis RtBphP1 undergoes red to far-red photoconversion with rapid thermal reversion to the dark state. RtBphP1 is autophosphorylated in the dark; this activity is inhibited under red light. The RtBphP1 cognate response regulator, the R. tataouinensis bacteriophytochrome response regulator (RtBRR), and a homolog, AtBRR from Agrobacterium tumefaciens, crystallize unexpectedly as arm-in-arm dimers, reliant on a conserved hydrophobic motif, hFWAhL (where h is a hydrophobic M, V, L, or I residue). RtBRR and AtBRR dimerize distinctly from four structurally characterized phytochrome response regulators found in photosynthetic organisms and from all other receiver domain homodimers in the Protein Data Bank. A unique cacodylate-zinc-histidine tag metal organic framework yielded single-wavelength anomalous diffraction phases and may be of general interest. Examination of the effect of the BRR stoichiometry on signal transduction showed that phosphorylated RtBRR is accumulated more efficiently than the engineered monomeric RtBRR (RtBRRmon) in phosphotransfer reactions. Thus, we conclude that arm-in-arm dimers are a relevant signaling intermediate in this class of two-component regulatory systems. BphP histidine kinases and their cognate response regulators comprise widespread red light-sensing two-component systems. Much work on BphPs has focused on structural understanding of light sensing and on enhancing the natural infrared fluorescence of these proteins, rather than on signal transduction or the resultant phenotypes. To begin to address this knowledge gap, we solved the crystal structures of two single-domain response regulators encoded by a region immediately downstream of that encoding BphPs. We observed a previously unknown arm-in-arm dimer linkage. Monomerization via deletion of the C-terminal dimerization motif had an inhibitory effect on net response regulator phosphorylation, underlining the importance of these unusual dimers for signal transduction.

TRF2-Mediated Control of Telomere DNA Topology as a Mechanism for Chromosome-End Protection

The shelterin proteins protect telomeres against activation of the DNA damage checkpoints and recombinational repair. We show here that a dimer of the shelterin subunit TRF2 wraps ∼ 90 bp of DNA through several lysine and arginine residues localized around its homodimerization domain. The expression of a wrapping-deficient TRF2 mutant, named Top-less, alters telomeric DNA topology, decreases the number of terminal loops (t-loops), and triggers the ATM checkpoint, while still protecting telomeres against non-homologous end joining (NHEJ). In Top-less cells, the protection against NHEJ is alleviated if the expression of the TRF2-interacting protein RAP1 is reduced. We conclude that a distinctive topological state of telomeric DNA, controlled by the TRF2-dependent DNA wrapping and linked to t-loop formation, inhibits both ATM activation and NHEJ. The presence of RAP1 at telomeres appears as a backup mechanism to prevent NHEJ when topology-mediated telomere protection is impaired.

Diverse oligomeric states of CEACAM IgV domains

Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) comprise a large family of cell surface adhesion molecules that bind to themselves and other family members to carry out numerous cellular functions, including proliferation, signaling, differentiation, tumor suppression, and survival. They also play diverse and significant roles in immunity and infection. The formation of CEACAM oligomers is caused predominantly by interactions between their N-terminal IgV domains. Although X-ray crystal structures of CEACAM IgV domain homodimers have been described, how CEACAMs form heterodimers or remain monomers is poorly understood. To address this key aspect of CEACAM function, we determined the crystal structures of IgV domains that form a homodimeric CEACAM6 complex, monomeric CEACAM8, and a heterodimeric CEACAM6-CEACAM8 complex. To confirm and quantify these interactions in solution, we used analytical ultracentrifugation to measure the dimerization constants of CEACAM homodimers and isothermal titration calorimetry to determine the thermodynamic parameters and binding affinities of CEACAM heterodimers. We found the CEACAM6-CEACAM8 heterodimeric state to be substantially favored energetically relative to the CEACAM6 homodimer. Our data provide a molecular basis for the adoption of the diverse oligomeric states known to exist for CEACAMs and suggest ways in which CEACAM6 and CEACAM8 regulate the biological functions of one another, as well as of additional CEACAMs with which they interact, both in cis and in trans.

Crystal structure of the G3BP2 NTF2-like domain in complex with a canonical FGDF motif peptide

The crystal structure of the NTF2-like domain of the human Ras GTPase SH3 Binding Protein (G3BP), isoform 2, was determined at a resolution of 2.75 Å in complex with a peptide containing a FGDF sequence motif. The overall structure of the protein is highly similar to the homodimeric N-terminal domains of the G3BP1 and Rasputin proteins. Recently, a subset of G3BP interacting proteins was recognized to share a common sequence motif, FGDF. The most studied binding partners, USP10 and viral nsP3, interfere with essential G3BP functions related to assembly of cellular stress granules. Reported molecular modeling suggested that FGDF-motif containing peptides bind in an extended conformation into a hydrophobic groove on the surface of the G3BP NTF2-like domain in a manner similar to the known binding of FxFG nucleoporin repeats. The results in this paper provide evidence for a different binding mode. The FGDF peptide binds and changes conformation of the protruding N-terminal residues by providing hydrophobic interactions to a symmetry related molecule that facilitated crystallization of the G3BP2 isoform.

Genome-wide binding and mechanistic analyses of Smchd1-mediated epigenetic regulation

Structural maintenance of chromosomes flexible hinge domain containing 1 (Smchd1) is an epigenetic repressor with described roles in X inactivation and genomic imprinting, but Smchd1 is also critically involved in the pathogenesis of facioscapulohumeral dystrophy. The underlying molecular mechanism by which Smchd1 functions in these instances remains unknown. Our genome-wide transcriptional and epigenetic analyses show that Smchd1 binds cis-regulatory elements, many of which coincide with CCCTC-binding factor (Ctcf) binding sites, for example, the clustered protocadherin (Pcdh) genes, where we show Smchd1 and Ctcf act in opposing ways. We provide biochemical and biophysical evidence that Smchd1-chromatin interactions are established through the homodimeric hinge domain of Smchd1 and, intriguingly, that the hinge domain also has the capacity to bind DNA and RNA. Our results suggest Smchd1 imparts epigenetic regulation via physical association with chromatin, which may antagonize Ctcf-facilitated chromatin interactions, resulting in coordinated transcriptional control.

Structural basis of natural promoter recognition by the retinoid X nuclear receptor.

Retinoid X receptors (RXRs) act as homodimers or heterodimerisation partners of class II nuclear receptors. RXR homo- and heterodimers bind direct repeats of the half-site (A/G)G(G/T)TCA separated by 1 nucleotide (DR1). We present a structural characterization of RXR-DNA binding domain (DBD) homodimers on several natural DR1s and an idealized symmetric DR1. Homodimers displayed asymmetric binding, with critical high-affinity interactions accounting for the 3′ positioning of RXR in heterodimers on DR1s. Differing half-site and spacer DNA sequence induce changes in RXR-DBD homodimer conformation notably in the dimerization interface such that natural DR1s are bound with higher affinity than an idealized symmetric DR1. Subtle changes in the consensus DR1 DNA sequence therefore specify binding affinity through altered RXR-DBD-DNA contacts and changes in DBD conformation suggesting a general model whereby preferential half-site recognition determines polarity of heterodimer binding to response elements.

In Vitro Reassembly of the Ribose ATP-binding Cassette Transporter Reveals a Distinct Set of Transport Complexes

Bacterial ATP-binding cassette (ABC) importers are primary active transporters that are critical for nutrient uptake. Based on structural and functional studies, ABC importers can be divided into two distinct classes, type I and type II. Type I importers follow a strict alternating access mechanism that is driven by the presence of the substrate. Type II importers accept substrates in a nucleotide-free state, with hydrolysis driving an inward facing conformation. The ribose transporter in Escherichia coli is a tripartite complex consisting of a cytoplasmic ATP-binding cassette protein, RbsA, with fused nucleotide binding domains; a transmembrane domain homodimer, RbsC2; and a periplasmic substrate binding protein, RbsB. To investigate the transport mechanism of the complex RbsABC2, we probed intersubunit interactions by varying the presence of the substrate ribose and the hydrolysis cofactors, ATP/ADP and Mg(2+). We were able to purify a full complex, RbsABC2, in the presence of stable, transition state mimics (ATP, Mg(2+), and VO4); a RbsAC complex in the presence of ADP and Mg(2+); and a heretofore unobserved RbsBC complex in the absence of cofactors. The presence of excess ribose also destabilized complex formation between RbsB and RbsC. These observations suggest that RbsABC2 shares functional traits with both type I and type II importers, as well as possessing unique features, and employs a distinct mechanism relative to other ABC transporters.

Molecular architecture of the ErbB2 extracellular domain homodimer.

Human epidermal growth factor receptors (HERs or ErbBs) play crucial roles in numerous cellular processes. ErbB2 is a key member of ErbB family, and its overexpression is recognized as a frequent molecular abnormality. In cancer, this overexpression correlates with aggressive disease and poor patient outcomes. Dimer-dependent phosphorylation is a key event for the signal transduction of ErbBs. However, the molecular mechanism of the dimerization of ErbB2 remains elusive. In the present work, we report the homodimer architecture of the ErbB2 extracellular domain (ECD) which is unique compared with other dimer-models of ErbBs. The structure of the ErbB2 ECD homodimer represents a "back to head" interaction, in which a protruding β-hairpin arm in domain II of one ErbB2 protomer is inserted into a C-shaped pocket created by domains I-III of the adjacent ErbB2 protomer. This dimerized architecture and its impact on the phosphorylation of ErbB2 intracellular domain were further verified by a mutagenesis study. We also elucidated the different impacts of two clinically administered therapeutic antibodies, trastuzumab and pertuzumab, on ErbB2 dimerization. This information not only provides an understanding of the molecular mechanism of ErbBs dimerization but also elucidates ErbB2-targeted therapy at the molecular level.

Conformational Changes and Complex Formation of the Non-Canonical Ribose ABC Transporter

Bacterial ATP-Binding Cassette (ABC) transporters are vital for nutrient uptake. Structural and functional studies have revealed two distinct types (I and II) of importers based primarily upon mechanistic differences. The ribose transporter (RbsABC) in E. coli is a tripartite ABC importer. RbsABC consists of a cytoplasmic ATP-binding cassette, RbsA, with dual fused nucleotide-binding domains (NBD), a homodimeric transmembrane domain (TMD), RbsC, and a periplasmic substrate binding protein (SBP), RbsB. This study, using Electron Paramagnetic Resonance (EPR) spectroscopy and biochemical methods, demonstrates that RbsABC is a non-canonical ABC transporter that shares some structural and functional features with both type I and type II importers. ATP hydrolysis in the NBD is stimulated by the presence of substrate-loaded SBP, leading to ribose release, which is typical of type I importers. Quantitative measurements obtained from site-directed spin-labeling EPR spectroscopy reveal that hydrolysis promotes the opening of RbsB to release ribose and subsequently RbsB. Post-hydrolysis, both nucleotide and magnesium are required to maintain the complex interactions of RbsC and RbsA, based upon co-purification studies and EPR experiments, which is atypical for ABC importers of either type, where the high-affinity TMD-NBD association is maintained perpetually. In addition, the presence of ribose has a destabilizing effect on RbsB-RbsC interactions, a type II importer behavior. Further, RbsB and RbsC interact in an unusual apo-complex consisting only of the SBP-TMD. Finally, substrate transport is accomplished by hydrolysis at a single consensus site in RbsA, with the second site rendered degenerate by mutations of conserved amino acids. This feature is not uncommon to ABC exporters, but heretofore unobserved in importers. Taking these observations together, RbsABC appears to transport substrate by a distinct mechanism, and offers an opportunity to gain insight into the function of non-canonical systems.

Functional characterization of the TERRA transcriptome at damaged telomeres.

Telomere deprotection occurs during tumorigenesis and aging upon telomere shortening or loss of the telomeric shelterin component TRF2. Deprotected telomeres undergo changes in chromatin structure and elicit a DNA damage response (DDR) that leads to cellular senescence. The telomeric long noncoding RNA TERRA has been implicated in modulating the structure and processing of deprotected telomeres. Here, we characterize the human TERRA transcriptome at normal and TRF2-depleted telomeres and demonstrate that TERRA upregulation is occurring upon depletion of TRF2 at all transcribed telomeres. TRF2 represses TERRA transcription through its homodimerization domain, which was previously shown to induce chromatin compaction and to prevent the early steps of DDR activation. We show that TERRA associates with SUV39H1 H3K9 histone methyltransferase, which promotes accumulation of H3K9me3 at damaged telomeres and end-to-end fusions. Altogether our data elucidate the TERRA landscape and defines critical roles for this RNA in the telomeric DNA damage response.

A SIRT7-Dependent Acetylation Switch of GABPβ1 Controls Mitochondrial Function

Mitochondrial activity is controlled by proteins encoded by both nuclear and mitochondrial DNA. Here, we identify Sirt7 as a crucial regulator of mitochondrial homeostasis. Sirt7 deficiency in mice induces multisystemic mitochondrial dysfunction, which is reflected by increased blood lactate levels, reduced exercise performance, cardiac dysfunction, hepatic microvesicular steatosis, and age-related hearing loss. This link between SIRT7 and mitochondrial function is translatable in humans, where SIRT7 overexpression rescues the mitochondrial functional defect in fibroblasts with a mutation in NDUFSI. These wide-ranging effects of SIRT7 on mitochondrial homeostasis are the consequence of the deacetylation of distinct lysine residues located in the hetero- and homodimerization domains of GABPβ1, a master regulator of nuclear-encoded mitochondrial genes. SIRT7-mediated deacetylation of GABPβ1 facilitates complex formation with GABPα and the transcriptional activation of the GABPα/GABPβ heterotetramer. Altogether, these data suggest that SIRT7 is a dynamic nuclear regulator of mitochondrial function through its impact on GABPβ1 function.