Shintaro Aibara

Development of Cell-Permeable, Non-Helical Constrained Peptides to Target a Key Protein-Protein Interaction in Ovarian Cancer.

Abstract There is a lack of current treatment options for ovarian clear cell carcinoma (CCC) and the cancer is often resistant to platinum‐based chemotherapy. Hence there is an urgent need for novel therapeutics. The transcription factor hepatocyte nuclear factor 1β (HNF1β) is ubiquitously overexpressed in CCC and is seen as an attractive therapeutic target. This was validated through shRNA‐mediated knockdown of the target protein, HNF1β, in five high‐ and low‐HNF1β‐expressing CCC lines. To inhibit the protein function, cell‐permeable, non‐helical constrained proteomimetics to target the HNF1β–importin α protein–protein interaction were designed, guided by X‐ray crystallographic data and molecular dynamics simulations. In this way, we developed the first reported series of constrained peptide nuclear import inhibitors. Importantly, this general approach may be extended to other transcription factors.

The principal mRNA nuclear export factor NXF1:NXT1 forms a symmetric binding platform that facilitates export of retroviral CTE-RNA

The NXF1:NXT1 complex (also known as TAP:p15) is a general mRNA nuclear export factor that is conserved from yeast to humans. NXF1 is a modular protein constructed from four domains (RRM, LRR, NTF2-like and UBA domains). It is currently unclear how NXF1:NXT1 binds transcripts and whether there is higher organization of the NXF1 domains. We report here the 3.4 Å resolution crystal structure of the first three domains of human NXF1 together with NXT1 that has two copies of the complex in the asymmetric unit arranged to form an intimate domain-swapped dimer. In this dimer, the linkers between the NXF1 LRR and NTF2-like domains interact with NXT1, generating a 2-fold symmetric platform in which the RNA-binding RRM, LRR and NTF2-like domains are arranged on one face. In addition to bulk transcripts, NXF1:NXT1 also facilitates the export of unspliced retroviral genomic RNA from simple type-D retroviruses such as SRV-1 that contain a constitutive transport element (CTE), a cis-acting 2-fold symmetric RNA stem–loop motif. Complementary structural, biochemical and cellular techniques indicated that the formation of a symmetric RNA binding platform generated by dimerization of NXF1:NXT1 facilitates the recognition of CTE-RNA and promotes its nuclear export.

Structures of the human mitochondrial ribosome in native states of assembly

Mammalian mitochondrial ribosomes (mitoribosomes) have less rRNA content and 36 additional proteins compared with the evolutionarily related bacterial ribosome. These differences make the assembly of mitoribosomes more complex than the assembly of bacterial ribosomes, but the molecular details of mitoribosomal biogenesis remain elusive. Here, we report the structures of two late-stage assembly intermediates of the human mitoribosomal large subunit (mt-LSU) isolated from a native pool within a human cell line and solved by cryo-EM to ∼3-Å resolution. Comparison of the structures reveals insights into the timing of rRNA folding and protein incorporation during the final steps of ribosomal maturation and the evolutionary adaptations that are required to preserve biogenesis after the structural diversification of mitoribosomes. Furthermore, the structures redefine the ribosome silencing factor (RsfS) family as multifunctional biogenesis factors and identify two new assembly factors (L0R8F8 and mt-ACP) not previously implicated in mitoribosomal biogenesis.

Structural Characterization of the Chaetomium thermophilum TREX-2 Complex and its Interaction with the mRNA Nuclear Export Factor Mex67:Mtr2

Summary The TREX-2 complex integrates mRNA nuclear export into the gene expression pathway and is based on a Sac3 scaffold to which Thp1, Sem1, Sus1, and Cdc31 bind. TREX-2 also binds the mRNA nuclear export factor, Mex67:Mtr2, through the Sac3 N-terminal region (Sac3N). Here, we characterize Chaetomium thermophilum TREX-2, show that the in vitro reconstituted complex has an annular structure, and define the structural basis for interactions between Sac3, Sus1, Cdc31, and Mex67:Mtr2. Crystal structures show that the binding of C. thermophilum Sac3N to the Mex67 NTF2-like domain (Mex67NTF2L) is mediated primarily through phenylalanine residues present in a series of repeating sequence motifs that resemble those seen in many nucleoporins, and Mlp1 also binds Mex67:Mtr2 using a similar motif. Deletion of Sac3N generated growth and mRNA export defects in Saccharomyces cerevisiae, and we propose TREX-2 and Mlp1 function to facilitate export by concentrating mature messenger ribonucleoparticles at the nuclear pore entrance.

Domain organization within the nuclear export factor Mex67:Mtr2 generates an extended mRNA binding surface

The Mex67:Mtr2 complex is the principal yeast nuclear export factor for bulk mRNA and also contributes to ribosomal subunit export. Mex67 is a modular protein constructed from four domains (RRM, LRR, NTF2-like and UBA) that have been thought to be joined by flexible linkers like beads on a string, with the RRM and LRR domains binding RNAs and the NTF2-like and UBA domains binding FG-nucleoporins to facilitate movement through nuclear pores. Here, we show that the NTF2-like domain from Saccharomyces cerevisiae Mex67:Mtr2 also contributes to RNA binding. Moreover, the 3.3 Å resolution crystal structure of the Mex67ΔUBA:Mtr2 complex, supplemented with small angle X-ray scattering data, indicated that the LRR domain has a defined spatial relationship to the Mex67NTF2L:Mtr2 region. Conversely, the RRM domain and especially the UBA domain are more mobile. The conformation assumed by the LRR and NTF2-like domains results in clusters of positively-charged residues on each becoming arranged to form a continuous interface for binding RNA on the opposite side of the complex to the region that interacts with FG-nucleoporins to facilitate passage through nuclear pores.

Structural basis for the dimerization of Nab2 generated by RNA binding provides insight into its contribution to both poly(A) tail length determination and transcript compaction in Saccharomyces cerevisiae.

Abstract In Saccharomyces cerevisiae generation of export-competent mRNPs terminates the nuclear phase of the gene expression pathway and facilitates transport to the cytoplasm for translation. Nab2 functions in this process to control both mRNP compaction that facilitates movement through nuclear pore complexes and the length of transcript poly(A) tails. Nab2 has a modular structure that includes seven CCCH Zn fingers that bind to A-rich RNAs and fingers 5–7 are critical for these functions. Here, we demonstrate, using both biophysical and structural methods, that binding A11G RNA induces dimerization of Zn fingers 5–7 mediated by the novel spatial arrangement of the fingers promoting each RNA chain binding two protein chains. The dimerization of Nab2 induced by RNA binding provides a basis for understanding its function in both poly(A) tail length regulation and in the compaction of mature transcripts to facilitate nuclear export.

WD40 domain of Apc1 is critical for the coactivator-induced allosteric transition that stimulates APC/C catalytic activity.

Significance The anaphase-promoting complex/cyclosome (APC/C) is a large E3 ubiquitin ligase that controls progression through mitosis and entry into G1. Its capacity to recognize and ubiquitinate substrates is dependent on coactivator subunits that interact with substrate degrons and promote a conformational change of the APC/C to increase its affinity for the priming E2 UbcH10. We show that the WD40 domain of anaphase-promoting complex subunit 1 (Apc1) is required for communicating the conformational change initiated by the binding of coactivator to the catalytic module. In contrast to UbcH10, binding of the elongating E2 Ube2S and its APC/C-stimulated activity does not require the active state of the APC/C. The work raises the possibility that conformational changes of the Apc1 WD40 domain may play a role in regulating UbcH10 binding to the APC/C. The anaphase-promoting complex/cyclosome (APC/C) is a large multimeric cullin–RING E3 ubiquitin ligase that orchestrates cell-cycle progression by targeting cell-cycle regulatory proteins for destruction via the ubiquitin proteasome system. The APC/C assembly comprises two scaffolding subcomplexes: the platform and the TPR lobe that together coordinate the juxtaposition of the catalytic and substrate-recognition modules. The platform comprises APC/C subunits Apc1, Apc4, Apc5, and Apc15. Although the role of Apc1 as an APC/C scaffolding subunit has been characterized, its specific functions in contributing toward APC/C catalytic activity are not fully understood. Here, we report the crystal structure of the N-terminal domain of human Apc1 (Apc1N) determined at 2.2-Å resolution and provide an atomic-resolution description of the architecture of its WD40 (WD40 repeat) domain (Apc1WD40). To understand how Apc1WD40 contributes to APC/C activity, a mutant form of the APC/C with Apc1WD40 deleted was generated and evaluated biochemically and structurally. We found that the deletion of Apc1WD40 abolished the UbcH10-dependent ubiquitination of APC/C substrates without impairing the Ube2S-dependent ubiquitin chain elongation activity. A cryo-EM structure of an APC/C–Cdh1 complex with Apc1WD40 deleted showed that the mutant APC/C is locked into an inactive conformation in which the UbcH10-binding site of the catalytic module is inaccessible. Additionally, an EM density for Apc15 is not visible. Our data show that Apc1WD40 is required to mediate the coactivator-induced conformational change of the APC/C that is responsible for stimulating APC/C catalytic activity by promoting UbcH10 binding. In contrast, Ube2S activity toward APC/C substrates is not dependent on the initiation-competent conformation of the APC/C.

Mechanistic Insights into Autoinhibition of the Oncogenic Chromatin Remodeler ALC1

Summary Human ALC1 is an oncogene-encoded chromatin-remodeling enzyme required for DNA repair that possesses a poly(ADP-ribose) (PAR)-binding macro domain. Its engagement with PARylated PARP1 activates ALC1 at sites of DNA damage, but the underlying mechanism remains unclear. Here, we establish a dual role for the macro domain in autoinhibition of ALC1 ATPase activity and coupling to nucleosome mobilization. In the absence of DNA damage, an inactive conformation of the ATPase is maintained by juxtaposition of the macro domain against predominantly the C-terminal ATPase lobe through conserved electrostatic interactions. Mutations within this interface displace the macro domain, constitutively activate the ALC1 ATPase independent of PARylated PARP1, and alter the dynamics of ALC1 recruitment at DNA damage sites. Upon DNA damage, binding of PARylated PARP1 by the macro domain induces a conformational change that relieves autoinhibitory interactions with the ATPase motor, which selectively activates ALC1 remodeling upon recruitment to sites of DNA damage.

Structural characterization of the principal mRNA-export factor Mex67–Mtr2 from Chaetomium thermophilum

The crystal structures of the individual domains of the Mex67–Mtr2 complex from C. thermophilum have been determined and their arrangement in solution has been studied by SAXS.

The Sac3 TPR-like region in the Saccharomyces cerevisiae TREX-2 complex is more extensive but independent of the CID region.

Transcription-export complex 2 (TREX-2 complex) facilitates the localization of actively transcribing genes to the nuclear periphery and also functions to contribute to the generation of export-competent mRNPs through interactions with the general mRNA nuclear export factor Mex67:Mtr2. The TREX-2 complex is based on a Sac3 scaffold to which Thp1, Sem1, Cdc31, and Sus1 bind. TREX-2 can be subdivided into two modules: one, in which Thp1 and Sem1 bind to the Sac3M region (residues ∼100–551), and the other in which Cdc31 and two Sus1 chains bind to the Sac3CID region (residues ∼710–805). Complementary structural analyses using X-ray crystallography, electron microscopy, and small-angle X-ray scattering of the Saccharomyces cerevisiae TREX-2 complex, expressed using Baculovirus-infected Sf9 cells, have indicated that the TPR-like repeats of the Sac3M region extend considerably further towards the N-terminus than previously thought, and also indicate that this region and Sac3CID:Sus1:Cdc31 region of the S. cerevisiae complex are structurally independent. Although the density visible accounted for only ∼100 kDa, a 5.3 Å resolution cryo-EM reconstruction was obtained of the M-region of TREX-2 that showed an additional three putative α-helices extending towards the Sac3 N-terminus and these helices were also seen in a 4.9 Å resolution structure obtained by X-ray crystallography. Summary statement We describe the expression, purification and structural characterization of the S. cerevisiae TREX-2 complex and demonstrate that the Sac3 TPR-like repeats are more extensive than previously thought and that the M- and CID-regions do not appear to have a defined spatial orientation.