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Human transthyretin (TTR) is implicated in several fatal forms of amyloidosis. Many mutations of TTR have been identified; most of these are pathogenic, but some offer protective effects. The molecular basis underlying the vastly different fibrillation behaviours of these TTR mutants is poorly understood. Here, on the basis of neutron crystallography, native mass spectrometry and modelling studies, we propose a mechanism whereby TTR can form amyloid fibrils via a parallel equilibrium of partially unfolded species that proceeds in favour of the amyloidogenic forms of TTR. It is suggested that unfolding events within the TTR monomer originate at the C-D loop of the protein, and that destabilising mutations in this region enhance the rate of TTR fibrillation. Furthermore, it is proposed that the binding of small molecule drugs to TTR stabilises non-amyloidogenic states of TTR in a manner similar to that occurring for the protective mutants of the protein. A number of disease-causing human transthyretin (TTR) mutations are known to lead to amyloid formation. Here the authors combine neutron crystallography, native mass spectrometry and modelling studies to characterize the T119M and S52P-TTR mutants, providing mechanistic insights into TTR amyloidosis.

The Mitochondrial Complex I Assembly (MCIA) complex is essential for the biogenesis of respiratory Complex I (CI), the first enzyme in the respiratory chain, which has been linked to Alzheimer’s disease (AD) pathogenesis. However, how MCIA facilitates CI assembly, and how it is linked with AD pathogenesis, is poorly understood. Here we report the structural basis of the complex formation between the MCIA subunits ECSIT and ACAD9. ECSIT binding induces a major conformational change in the FAD-binding loop of ACAD9, releasing the FAD cofactor and converting ACAD9 from a fatty acid β-oxidation (FAO) enzyme to a CI assembly factor. We provide evidence that ECSIT phosphorylation downregulates its association with ACAD9 and is reduced in neuronal cells upon exposure to amyloid-β (Aβ) oligomers. These findings advance our understanding of the MCIA complex assembly and suggest a possible role for ECSIT in the reprogramming of bioenergetic pathways linked to Aβ toxicity, a hallmark of AD. The role that cellular bioenergetics plays in the early stages of Alzheimer’s disease is poorly understood. Here the authors describe structures of key OXPHOS assembly proteins, providing insights into how these pathways are interlinked and regulated.

The cohesin ring complex is required for numerous chromosomal transactions including sister chromatid cohesion, DNA damage repair and transcriptional regulation. How cohesin engages its chromatin substrate has remained an unresolved question. We show here, by determining a crystal structure of the budding yeast cohesin HEAT-repeat subunit Scc3 bound to a fragment of the Scc1 kleisin subunit and DNA, that Scc3 and Scc1 form a composite DNA interaction module. The Scc3-Scc1 subcomplex engages double-stranded DNA through a conserved, positively charged surface. We demonstrate that this conserved domain is required for DNA binding by Scc3-Scc1 in vitro, as well as for the enrichment of cohesin on chromosomes and for cell viability. These findings suggest that the Scc3-Scc1 DNA-binding interface plays a central role in the recruitment of cohesin complexes to chromosomes and therefore for cohesin to faithfully execute its functions during cell division.

Transcriptional factors ETS1/2 and p52 synergize downstream of non-canonical NF-κB signaling to drive reactivation of the −146C>T mutant TERT promoter in multiple cancer types, but the mechanism underlying this cooperativity remains unknown. Here we report the crystal structure of a ternary p52/ETS1/−146C>T TERT promoter complex. While p52 needs to associate with consensus κB sites on the DNA to function during non-canonical NF-κB signaling, we show that p52 can activate the −146C>T TERT promoter without binding DNA. Instead, p52 interacts with ETS1 to form a heterotetramer, counteracting autoinhibition of ETS1. Analogous to observations with the GABPA/GABPB heterotetramer, the native flanking ETS motifs are required for sustained activation of the −146C>T TERT promoter by the p52/ETS1 heterotetramer. These observations provide a unifying mechanism for transcriptional activation by GABP and ETS1, and suggest that genome-wide targets of non-canonical NF-κB signaling are not limited to those driven by consensus κB sequences. Incessant telomere synthesis in cancer cells depends on specific mutations in the TERT promoter, enabling its activation by transcription factors ETS1 and p52. Here, the authors elucidate the structural basis for p52/ETS1 binding to mutant TERT , suggesting a general mechanism for TERT reactivation in cancer.

Dear Editor, The survival of motor neurons （SMN） complex assembles the heptameric rings of Sm proteins （Sm core） on small nuclear RNAs （snRNAs） to form small nuclear ribonucleoprotein particles （snRNPs） [1-4], the major components of the spliceosome [5]. The SMN complex consists of SMN, Unrip and Gemin2-8 [6, 7]. SMN deficiency leads to correspondingly reduced snRNP assembly and is an underlying cause for spinal muscular atrophy, a common motor neuron degenerative disease [8]. The SMN complex functions to ensure Sm core assembly only on the correct snRNAs, therefore preventing aberrant Sm core formation. Such stringent specificity of the SMN complex toward snRNAs is conferred by Gemin5, which directly interacts with sn- RNA precursors （pre-snRNAs） [9-11] and delivers them to sites of Sm core assembly and processing. Gemin5 recognizes the snRNP code comprised of the Sm site （AU5-6G） and an adjacent Y-terminal stem loop via its N-terminal WD40 repeat domain （termed Gemin5-WD）. Gemin5 also has been shown to bind the 7-methylguanosine （m7G） cap [12] and to downregulate internal ribosome entry site-dependent translation [13]. Although the function of Gemin5 in the biogenesis of snRNPs has been explored, the molecular mechanism for specific recognition of pre-snRNAs by Gemin5 remains elusive.

Non-ribosomal peptide synthetases (NRPS) are multi-modular/domain enzymes that catalyze the synthesis of bioactive peptides. A crucial step in the process is peptide elongation accomplished by the condensation (C) domain with the aid of a peptidyl carrier or thiolation (T) domain. Here, we examined condensation reaction carried out by NRPS AmbB involved in biosynthesis of l -2-amino-4-methoxy-trans-3-butenoic acid (AMB) in P. aeruginosa . We determined crystal structures of the truncated T–C bidomain of AmbB in three forms, the apo enzyme with disordered T domain, the holo form with serine linked phosphopantetheine (Ppant) and a holo form with substrate ( l -alanine) loaded onto Ppant. The two holo forms feature the T domain in a substrate-donation conformation. Mutagenesis combined with functional assays identified residues essential for the attachment of Ppant, anchoring the Ppant- l -Ala in the donor catalytic channel and the role of the conserved His953 in condensation activity. Altogether, these results provide structural insights into the condensation reaction at the donor site with a substrate-bound C domain of AmbB and lay the foundation for understanding the molecular mechanism of condensation which is crucial for AMB synthesis.

Correct 3’end processing of mRNAs is one of the regulatory cornerstones of gene expression. In a parasite that must adapt to the regulatory requirements of its multi-host life style, there is a need to adopt additional means to partition the distinct transcriptional signatures of the closely and tandemly arranged stage-specific genes. In this study, we report our findings in T. gondii of an m6A-dependent 3’end polyadenylation serving as a transcriptional barrier at these loci . We identify the core polyadenylation complex within T. gondii and establish CPSF4 as a reader for m6A-modified mRNAs, via a YTH domain within its C-terminus, a feature which is shared with plants. We bring evidence of the specificity of this interaction both biochemically, and by determining the crystal structure at high resolution of the T. gondii CPSF4-YTH in complex with an m6A-modified RNA. We show that the loss of m6A, both at the level of its deposition or its recognition is associated with an increase in aberrantly elongated chimeric mRNAs emanating from impaired transcriptional termination, a phenotype previously noticed in the plant model Arabidopsis thaliana . Nanopore direct RNA sequencing shows the occurrence of transcriptional read-through breaching into downstream repressed stage-specific genes, in the absence of either CPSF4 or the m6A RNA methylase components in both T. gondii and A. thaliana . Taken together, our results shed light on an essential regulatory mechanism coupling the pathways of m6A metabolism directly to the cleavage and polyadenylation processes, one that interestingly seem to serve, in both T. gondii and A. thaliana , as a guardian against aberrant transcriptional read-throughs.

β-Phosphoglucomutase (βPGM) catalyzes isomerization of β- d -glucose 1-phosphate (βG1P) into d -glucose 6-phosphate (G6P) via sequential phosphoryl transfer steps using a β- d -glucose 1,6-bisphosphate (βG16BP) intermediate. Synthetic fluoromethylenephosphonate and methylenephosphonate analogs of βG1P deliver novel step 1 transition state analog (TSA) complexes for βPGM, incorporating trifluoromagnesate and tetrafluoroaluminate surrogates of the phosphoryl group. Within an invariant protein conformation, the β- d -glucopyranose ring in the βG1P TSA complexes (step 1) is flipped over and shifted relative to the G6P TSA complexes (step 2). Its equatorial hydroxyl groups are hydrogen-bonded directly to the enzyme rather than indirectly via water molecules as in step 2. The (C)O–P bond orientation for binding the phosphate in the inert phosphate site differs by ∼30° between steps 1 and 2. By contrast, the orientations for the axial O–Mg–O alignment for the TSA of the phosphoryl group in the catalytic site differ by only ∼5°, and the atoms representing the five phosphorus-bonded oxygens in the two transition states (TSs) are virtually superimposable. The conformation of βG16BP in step 1 does not fit into the same invariant active site for step 2 by simple positional interchange of the phosphates: the TS alignment is achieved by conformational change of the hexose rather than the protein.